(A) Impaired cell accumulation. The number of OT-I cells remaining after transfer into Act-mOVA–mated females was calculated as a percentage of total CD8⁺ T cells, then normalized to the number of cells remaining after equivalent time periods in B6-mated mice injected in parallel. Similarly, the number of OT-I cells remaining in virgin females treated with OVA or OVA plus anti-CD40 antibodies and poly(I:C) was normalized to the number remaining in untreated virgins. Each point represents mean ± SEM of n = 3–10 Act-mOVA–mated or n = 2–3 virgin mice. (B) Impaired modulation of CD62L and CD25 expression. OT-I cells were analyzed either 7 days after transfer into E10.5 Act-mOVA–mated or in virgin mice 3 days after treatment with OVA plus anti-CD40 antibodies and poly(I:C). Plots are representative of 3 independent experiments, each with individual mice or pools of n = 2–5 Act-mOVA–mated and n = 1–3 virgin mice per experiment. Plots display similar cell numbers in the 2 groups of mice, even though this does not represent their relative prevalence in vivo. The percentage of OT-I cells with altered marker expression is shown. (C and D) Impaired cytokine expression. Seven days after transfer into E10.5 pregnant mice, OT-I cells in the spleen (C) or uterine LNs (D) were stained for intracellular cytokines. Virgin females were similarly analyzed 7 days after indicated treatments given at the time of OT-I transfer. The percentage of OT-I cells with a CFSE^locytokine⁺ phenotype is shown. Data are representative of 2 independent experiments, each with a pool of n = 4 Act-mOVA–mated mice. The other treatment groups show data representative of individual mice or pools encompassing a total of n = 2–6 mice per group.